Process for the synthesis of peptides containing a 4-hydroxy-proline substructure

ABSTRACT

The present invention relates to processes for preparing peptides and to intermediates involved in such processes, e.g. a process for preparing a compound of formula VIII 
     
       
         
         
             
             
         
       
     
     wherein R 12  and R 13  are as defined herein.

The present invention relates to processes for preparing peptides andintermediates involved in such processes.

In one aspect, the invention relates to:

(A) a process for preparing a compound of formula I

wherein R₁ is a reactive substituent or an attachment to a solid phase;

R₂ is a reactive substituent; and

R₃, R₄ and R₅ are each independently hydrogen or one or moresubstituents attached to each benzene ring and selected from hydroxy,amino, C₁₋₁₀-alkyl, C₁₋₁₀-alkoxy, C₁₋₁₀-alkylamino, di-C₁₋₁₀-alkylamino,carbamoyl, C₁₋₁₀-alkylcarbamoyl, di-C₁₋₁₀-alkylcarbamoyl,halo-C₁₋₁₀-alkyl, halogeno and nitro;

in free or salt form; comprising

(a) reacting a compound of formula VI with an electrophile:

wherein R₃, R₄ and R₅ are as defined above;

R₉ is —OH, —OM or —OMX, where M is metal and X is a nucleophilicsubstituent;

R₁₀ is -M or -MX, where M is metal and X is a nucleophilic substituent;in free or salt form;

and hydrolyzing the resulting compound to form a compound of formula Iwherein R₂ is hydroxy;

(b) optionally converting a compound of formula I wherein R₂ is hydroxyto a compound of formula I wherein R₂ is other than hydroxy;

(c) optionally converting R₁ in a compound of formula I to analternative R₁ group;

(d) optionally deprotecting a compound of formula I in protected form;and

(e) where required, converting a compound of formula I obtained in freeform into the desired salt form, or vice versa;

(B) a process for the preparation of a solid phase support system,comprising preparing a compound of formula I by a process as definedabove, and coupling the compound with a suitably derivatised orfunctionalised solid phase material;

(C) a compound of formula V in free or salt form

wherein R₃, R₄, R₅, and R₉ are as defined above; and

R₇ is a nucleophilic substituent;

(D) a compound of formula VI in free or salt form

wherein R₃, R₄, R₅ and R₉ are as defined above; and

R₁₀ is -M or -MX, where M is metal and X is a nucleophilic substituent.

The present invention provides a simple route for the preparation ofcompounds of formula I, which are useful for solid phase chemicalsynthesis. The process of the invention may directly produce a compoundof formula I attached to a solid phase, or where R₁ is a reactivesubstituent, the compound of formula I can easily be coupled to a solidphase at a later stage. The presence of the reactive substituent R₂permits the use of the compounds of formula I as linkers in thesynthesis of oligomers and polymers, such as glycopeptides, nucleotidesand proteins, especially in the solid phase synthesis of peptides. Thecompounds of formula I, particularly where R₂ is halogeno, may also beused as protecting agents for protecting functional groups, e.g. aminoor hydroxy groups, in chemical synthesis.

The compounds of formula V and VI are useful as intermediate compoundsin the preparation of compounds of formula I.

A compound of formula VI may be prepared by reacting a compound offormula V with a metal or organometallic compound:

wherein R₃, R₄, R₆ and R₉ are as defined above; and

R₇ is a nucleophilic substituent.

A compound of formula V may be prepared by:

(i) reacting a compound of formula II with a metal or organometalliccompound

wherein R₆ and R₇ are each a nucleophilic substituent and R₃ is asdefined above and is protected if necessary by a removable protectinggroup; and

(ii) reacting the compound obtained in (i) with a compound of formulaIII

wherein R₄ and R₅ are as defined above and are protected if necessary bya removable protecting group.

The process of the present invention may suitably be performed in asingle reaction vessel without intermediary isolation.

Terms used in the specification have the following meanings:

“Alkyl” may be straight or branched. Preferably alkyl is C₁₋₄alkyl.

“Alkoxy” may be straight or branched alkoxy. Preferably alkoxy isC₁₋₄alkoxy.

“Acylamino” denotes a group of formula —NH—C(O)—R where R is straightchain or branched C₁₋₁₀-alkyl, cycloalkyl or aryl. Preferably R isC₁₋₄alkyl.

“Acyloxy” denotes a group of formula —O—C(O)—R where R is as definedabove.

“Aryl” is preferably C₆₋₁₀ aryl, e.g. phenyl.

“Halogeno” means fluoro, chloro, bromo or iodo.

“Haloalkyl” means straight chain or branched C₁₋₄₀-alkyl, substituted byone or more, for example one, two or three halogen atoms, preferablyfluorine or chlorine atoms. Preferably haloalkyl is C₁₋₄-alkylsubstituted by one, two or three fluorine or chlorine atoms.

“Organometallic compound” denotes a compound in which a carbon atom ofan organic group is bound to a metal. The organometallic compound ispreferably an alkylmetallic compound, for example an alkyllithium, e.g.a straight or branched chain C₁₋₁₀ alkyllithium compound or mayalternatively be an arylmetallic compound, for example an arylithium.

More preferably the alkyllithium compound is a C₃₋₆ alkyllithiumcompound, such as butyllithium or hexyllithium.

Alternatively, the organometallic compound may be an organomagnesiumcompound, for example a straight or branched chain alkylmagnesium orarylmagnesium compound, preferably a C₁₋₆ alkylmagnesium compound.Organomagnesium compounds are commonly known as Grignard reagents. Theorganomagnesium compound is preferably an organomagnesium halide,especially an iodide or bromide.

In further alternative embodiments, the organometallic compound may bean alkyl- or arylzinc compound, for example a C₁₋₆-alkylzinc compound,or an C₁₋₄-alkyl- or aryltin compound.

M is preferably lithium or magnesium.

R₁ may be a reactive substituent suitable for linking the compound to asolid phase. R₁ may suitably be —C(O)R′, —C(O)—OR′, —C(O)—NR′R″,—R₁₂—NR′R″, —R₁₂—OR′, —NR′R″, or —C(O)X, wherein R′ and R″ are eachindependently hydrogen or straight or branched C₁₋₁₀-alkyl, e.g.C₁₋₄-alkyl, R₁₂ is straight or branched C₁₋₁₀-alkyl, e.g. C₁₋₄-alkyl,and X is a nucleophilic substituent, preferably halogeno, e.g. chloro.R₁ may suitably be in the para, ortho or meta position, preferably inthe para position.

Alternatively R₁ may be an attachment to a solid phase material, e.g.polystyrene. Preferably the attachment is of the formula —C(O)—P,—C(O)—OP, —C(O)—NR′—P, —R₁₂—NR′—P, —R₁₂—OP, —NR′—P, —C(O)—R₁₂—P,—C(O)—OR₁₂—P, —C(O)—NR′—R₁₂—P, —R₁₂—NR′—R₁₂—P, —R₁₂—OR₁₂—P, —NR′—R₁₂—Por —R₁₂—P, wherein R′, R″ and R₁₂ are as defined above and P is a solidphase material. More preferably R₁ is —C(O)—OP, —C(O)—OR₁₂—P,—C(O)—NH—P, —C(O)—NH—R₁₂—P, —NH—R₁₂—P or —R₁₂—P, wherein R₁₂ is methyl,e.g. —CH₂—P.

R₂ is preferably a reactive substituent suitable for linking thecompound to a biological oligomer or polymer, or a monomer unit thereof,e.g. an amino acid or polypeptide. R₂ may suitably be hydroxy,acylamino, acyloxy, amino, halogeno, sulfhydryl, C₁₋₁₀-alkoxy orC₆₋₄₀-aryloxy, preferably halogeno.

Each benzene ring shown in formulae I to VII may be substituted by oneor more groups. For example R₃ may designate one to four substituentgroups, preferably one or two substituent groups, attached to thebenzene ring shown in formulae I, II and IV to VII. R₄ and R₆ maydesignate one to five substituent groups, preferably one to threesubstituent groups, attached to each of the benzene rings shown informulae I, II and IV to VII. Each substituent group may be present atany suitable position on the benzene rings to which they are attached.More preferably R₄ and/or R₅ is a substituent group at the ortho or paraposition on the benzene ring to which it is attached.

Each of R₃, R₄ and R₅ may be protected by a removable protecting groupif necessary, e.g. when it contains an —OH or —NH₂ group which does notparticipate in the reaction. Protecting groups, their introduction andremoval are described, for example, In “Protective Groups in OrganicSynthesis”, T. W. Greene et al., John Wiley & Sons Inc., Second Edition1991. Preferably each of R₃, R₄ or R₅ is a group which does not requireprotection, e.g any of the groups listed above other than hydroxy, aminoor nitro.

When R₃, R₄ or R₅ is halogeno, it is preferably fluoro or chloro. WhenR₃, R₄ or R₅ is haloalkyl it is preferably trifluoromethyl. PreferablyR₃ is C₁₋₄-alkyl, halogeno, or hydrogen. Preferably R₄ and R₅ are eachindependently C₁₋₄-alkylcarbamoyl, dl-C₁₋₄-alkylcarbamoyl, carbamoyl,trifluoromethyl, fluoro or chloro. Preferably R₄ and R₅ are the same.

Preferably the nucleophilic substituents R₆ and R₇ are eachindependently halogeno, more preferably bromo or iodo, and mostpreferably R₆ and R₇ are each bromo. R₇ may suitably be in the para,ortho or meta position, preferably in the para position.

In one embodiment of the invention, the compound of formula II is firstreacted with the metal or organometallic compound to form a compound offormula IV:

wherein R₃ and R₇ are as defined above and R₈ is -M or -MX, where M ismetal and X is a nucleophilic substituent, preferably halogeno.

Where the metal is lithium or the organometallic compound is anorganolithium compound, R₈ is —Li. Where the metal is magnesium or theorganometallic compound is a Grignard reagent, R₈ is —MgX, and X ispreferably halogeno. The compound of formula IV is then reacted with acompound of formula III to form a compound of formula V.

The compounds of formulae IV and V need not be separated or isolated butmay be prepared in situ.

Suitable electrophiles for use in the process include carbon dioxide,isocyanates, nitrites, acyl halides (such as phosgene), leading to theformation of, for example, compounds of formula I wherein R₁ is carboxy,carbamoyl, alkylcarbamoyl or acyl. Alternatively the electrophile may bea derivatised solid phase material, e.g. a Merrifield polymer, enablingdirect coupling of the compound of formula VI to a solid phase. In oneembodiment the electrophile is a compound of formula X′—(CH₂)_(n)—P,wherein X′ is a nucleophilic substituent e.g. halogeno or tosyloxy, n isan Integer between 1 and 4, preferably 1, and P is a solid phasematerial.

Where the electrophile is carbon dioxide, the process preferablycomprises first reacting the compound of formula V with a metal ororganometallic compound to form a compound of formula VI as definedabove and reacting, preferably in situ, the compound of formula VI withcarbon dioxide.

Where the electrophile is carbon dioxide, preferably a compound offormula VII is formed:

wherein R₃, R₄, R₅ and R₉ are as defined above; and

R₁₁ is —OH, —OM or —OMX, where M is metal and X is a nucleophilicsubstituent, preferably halogeno, in salt or free form.

Alternatively, the carboxylation step comprises reacting a compound offormula V with carbon dioxide in the presence of a metal ororganometallic compound, to form a compound of formula VII.

The hydrolysis step preferably comprises reacting a compound of formulaVII wherein R₁₁ is —OM or —OMX and/or R₉ is —OM or —OMX with water or anacid yielding a compound of formula I wherein R₁ is carboxy and R₂ ishydroxy, in salt or free form. Suitable acids include ammonium chloride,acetic acid, sulphuric acid and hydrochloric acid. A pH-bufferedsolution may also be used. Preferably a weak acid is used and/or thestep may be carried out at a pH of 4 to 7. The reaction temperature mayconveniently be −50 to 50° C., preferably −10 to 10° C.

Alternatively the compound of formula VII where R₁₁ is —OM or —OMX maybe reacted with a a nucleophile, e.g. an amine or halide, to form acompound of formula I wherein R₁ is —C(O)—NR′R″ or —(O)—X and R′, R″ andX are as defined above.

The process of the invention may conveniently be carried out in an inertorganic solvent, preferably an ether solvent, for example diethyl ether,tetrahydrofuran or tert-butyl methyl ether. Alternatively a hydrocarbonsolvent may be used. The reaction temperature for step (a) isconveniently −30 to +10° C., preferably −5 to −0° C. The reaction may,for example, be carried out using 0.5 to 2 equivalents, preferably 0.8to 1.2 equivalents and most preferably about 1 equivalent of the metalor organometallic compound per equivalent of the compound of formula II.0.5 to 2 equivalents, preferably 0.8 to 1.2 equivalents of the compoundof formula III may be used per equivalent of the compound of formula II.

The temperature during the reaction of a compound of formula V with ametal or organometallic compound may conveniently be 0 to +50° C.,preferably +20 to +30° C. The reaction temperature for the reaction withan electrophile (e.g. CO₂) may conveniently be 0 to −30° C., preferably−5 to −10° C. The hydrolysis step, e.g. with acid, may conveniently beperformed at −10° C. to +10° C., e.g. 0 to +5° C. Preferably 0.5 to 2equivalents, more preferably 0.8 to 1.2 equivalents of a metal ororganometallic compound per equivalent of the compound of formula V areused.

The groups R₁ and R₂ may be converted to alternative R₁ and R₂ groupsspecified above by standard processes, such as by esterification,amidation or nucleophilic substitution. For example, a compound offormula I wherein R₂ is hydroxy may be converted to a compound offormula I wherein R₂ is halogeno by reaction with an acyl halide, e.g.acyl chloride.

Preferably the compound of formula I is in free form. The compounds infree or salt form can be obtained in the form of hydrates or solvatescontaining a solvent used for crystallization.

Compounds of formula I can be recovered from the reaction mixture andpurified in a conventional manner.

The starting compounds of formula II or formula III are known or may beprepared by methods analogous to those known in the art. Organometalliccompounds may be prepared by standard processes, for example by reactionof an alkyl or aryl halide with a metal, for example lithium ormagnesium, suspended in diethyl ether or tetrahydrofuran. Theorganometallic compound is preferably prepared and used in an inert(oxygen-free) anhydrous atmosphere, for instance under nitrogen.

The process according to the invention may suitably include a furtherstep of coupling the compound of formula I wherein R₁ is a reactivesubstituent to a solid phase material. Suitable solid phase materialsare disclosed, for example, in DE 4306839 A1, and include naturallyoccurring or synthetic organic or inorganic polymers in particulateform, e.g. as beads, or preferably as a surface layer on a suitableinert substrate material. Examples of suitable polymer materials includecrosslinked polystyrene, e.g. polystyrene pins, Gly-HMD-MA/DMA pins andHEMA pins. The compound of formula I may conveniently be coupled to asolid phase material by reacting a group present on the solid phase withR₁. Thus the solid phase material preferably comprises reactive groups,such as amino groups. Preferably a compound of formula I, wherein R₁ isa carboxy group or an activated carboxy group, e.g. by reaction withdiisopropylcarbodiimide, is reacted with a polymer bearing free aminogroups.

A compound of formula I may be used as a linker. Thus the processaccording to the invention may also suitably include a further step ofcoupling the compound of formula I, optionally bound to a solid phasematerial, to a biological oligomer or polymer, or a monomer unitthereof. The compound may conveniently be coupled to the biologicalmolecule, e.g. an amino acid or polypeptide, by reacting a group presenton the biological molecule with R₂. For example, where R₂ is hydroxy andthe biological molecule is a polypeptide or amino acid, the terminalcarboxylic acid group of the biological molecule can be esterified bythe R₂ hydroxy group, optionally via initial reaction of the compound offormula I with an acyl halide leading to in situ substitution of hydroxyby halogeno.

In a further aspect, the present invention provides:

(E) a process for preparing a compound of formula VIII

wherein R₁₂ and R₁₃ are each a removable protecting group and R₁₂ andR₁₃ are different; comprising reacting a compound of formula IX

with a suitable R₁₂ donor compound;

(F) intermediates useful in the above process, defined by the generalformula XIV

wherein R₁₆ is a removable protecting group other thanfluorenylmethoxycarbonyl, and is different to R₁₈;

R₁₇ is hydrogen or a blocking group removable by hydrolysis orhydrogenolysis; and

R₁₈ is hydrogen or a removable protecting group other thanfluorenylmethoxycarbonyl.

The present invention provides a simple and efficient route for thepreparation of compounds of formula VIII, which are useful in thesynthesis of peptides, for example as described in WO 02/10192. Thecompounds of formula XIV are useful as intermediate compounds in thepreparation of compounds of formula VIII.

The compound of formula IX may be prepared from a compound of formula X

wherein R₁₃ is as defined above,

R₁₄ is a removable protecting group and R₁₄ is different to R₁₂ and R₁₃,and

R₁₅ is a blocking group removable by hydrolysis or hydrogenolysis.

Protecting groups, their introduction and removal are described, forexample, in “Protective Groups in Organic Synthesis”, T. W. Greene etal., John Wiley & Sons Inc., Second Edition 1991. Suitable protectinggroup donor compounds, e.g. amino group protecting agents, arewell-known to a skilled person, e.g. anhydrides, halides, carbamates orN-hydroxysuccinimides which provide one of the protecting groups below.

The protecting group R₁₂ is preferably fluorenylmethoxycarbonyl. R₁₃ orR₁₆ is preferably a protecting group other thanfluorenylmethoxycarbonyl, and is preferably more resistant to removal byhydrolysis (for example base-catalysed hydrolysis) and/or hydrogenolysisthan R₁₂ and/or R₁₄, e.g. more resistant than fluorenylmethoxycarbonyland/or benzyloxycarbonyl. More preferably R₁₃ or R₁₆ istert-butoxycarbonyl.

The protecting group R₁₄ or R₁₈ is preferably more resistant to removalby hydrolysis than R₁₂, e.g. more resistant thanfluorenylmethoxycarbonyl. R₁₄ or R₁₈ is preferably removable byhydrogenolysis. Suitable R₁₄ or R₁₈ substituents includebenzyloxycarbonyl, 1,1,-dimethylpropynyloxycarbonyl, vinyloxycarbonyl,N-hydroxypiperidinyloxycarbonyl, 9-anthrylmethyloxycarbonyl andphenylaminothiocarbonyl, allyl, nitrobenzyl, triphenylmethyl,(p-methoxyphenyl)diphenylmethyl, diphenyl-4-pyridylmethyl orbenzylsulfonyl. Preferably R₁₄ or R₁₈ is an oxycarbonyl-containingprotecting group, e.g. benzyloxycarbonyl (carbobenzoxy).

R₁₅ or R₁₇ may suitably be:

(i) C₁₋₁₀-alkyl, e.g. C₁₋₄-alkyl, preferably methyl, ethyl, propyl orbutyl other than tert-butyl, more preferably methyl.

(ii) C₃₋₈-cycloalkyl, optionally substituted by one or more C₁₋₄ alkyl,e.g. methyl. Preferably cycloalkyl is C₃₋₆-cycloalkyl.

(iii) C₆₋₁₀-aryl, optionally substituted by one or more stabilisingsubstitutents, e.g halogeno or nitro. Preferably aryl is phenyl,optionally substituted by one, two or three halogeno, e.g. chloro.

(iv) (C₆₋₁₀-aryl)₁₋₃-C₁₋₁₀-alkyl, optionally substituted on the arylgroup by (I) one or more stabilising substituents, e.g halogeno ornitro, or (ii) by two substituents which together with the ring carbonatoms to which they are attached form a 5- or 6-membered ring,optionally containing one or two nitrogen or oxygen atoms.(C₆₋₁₀-aryl)₁₋₃-C₁₋₁₀-alkyl is preferably (I) (phenyl)₁₋₃-C₁₋₄-alkyl,more preferably benzyl, diphenylmethyl or triphenylmethyl, optionallysubstituted on each benzene ring by one, two or three halogeno, e.gchloro, (ii) anthrylmethyl, e.g. 9-anthrylmethyl, or (iii) piperonyl.

(v) C₆₋₁₀-aryl-C₁₋₄alkoxy-C₁₋₄-alkyl, preferably benzyloxymethyl.

(vi) C₆₋₁₀-aryl-carbonyl-C₁₋₄-alkyl, preferably phenacyl.

Preferably R₁₅ or R₁₇ is a group which is removable by hydrogenolysis,such as benzyl, benzyloxymethyl, phenacyl, triphenylmethyl, piperonyl or9-anthrylmethyl, preferably benzyl.

The compound of formula IX may be prepared by (i) hydrolysing the estercompound of formula X to obtain the corresponding carboxylic acid and(II) removing the protecting group R₁₄. Preferably the hydrolysis stepis performed before removal of the protecting group R₁₄. The protectinggroup R₁₄ may conveniently be removed by reductive hydrogenation(hydrogenolysis). This route, involving a hydrolysis step, is suitablyfollowed when R₁₅ is not removable by hydrogenolysis. The hydrolysisstep is preferably a base-catalysed hydrolysis, for example using sodiumhydroxide and may suitably be performed in a polar solvent, e.g.methanol.

Alternatively, a compound of formula IX may conveniently be prepared byhydrogenation (hydrogenolysis) of a compound of formula X wherein R₁₅ isa group which is removable by hydrogenolysis, e.g. benzyl. Thehydrogenation step may conveniently be performed using a suitablecatalytic agent, for instance palladium-on-charcoal.

Compound of formula X may be prepared by reacting a compound of formulaXI

wherein X is a nucleophilic substituent and R₁₄ and R₁₅ are as definedabove, with a compound of formula XII

wherein R₁₃ is as defined above. This step may be performed in anysuitable organic solvent, preferably in a hydrocarbon solvent, morepreferably toluene.

The compound of formula XII is protected ethylenediamine, wherein oneamino group has been protected with a removable protecting group. Thenucleophilic substituent X in formula XI is preferably halogeno, such asfluoro, chloro, bromo or iodo, more preferably chloro. The compound offormula XI wherein X is halogeno may be formed by reaction of a compoundof formula XIII

with an acyl halide, for instance phosgene, tri-phosgene,phenylchloroformate or 4-nitrophenylchloroformate, preferably4-nitrophenylchloroformate. This step may suitably be performed in thepresence of an organic base, e.g. dimethylaminopyridine, in a non-polarsolvent, e.g. toluene.

The compound of formula XIII may be commercially available, e.g. whenR₁₅ is methyl or may be formed by esterification of 4-hydroxy-prolineaccording to methods known in the art, for instance by reaction withbenzyl alcohol or methanol. The resulting ester is then protected byreaction with a suitable R₁₄ donor compound, e.g.benzyloxycarbonyl-N-hydroxysuccinimide.

The compound of formula XI need not be separated or isolated, as thecompound of formula XIII may be reacted with an acyl halide and theproduct of this reaction subsequently reacted with a compound of formulaXII in the same vessel.

The addition of the protecting group R₁₂ to the compound of formula IXmay suitably be performed in the presence of sodiumcarbonate/acetonitrile.

Compounds of formula VIII can be recovered from the reaction mixture andpurified in a conventional manner.

In the compounds of formulae VIII-XI and XIII above, the oxy substituenton the proline may be in position cis or trans, preferably trans. Thecis or trans isomers may be individually prepared, using thecorresponding cis or trans hydroxyproline as starting material.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as described thereafter.

In a further aspect, the present invention relates to a process forproducing a compound of formula VIII, wherein R₁₂ isfluorenylmethoxycarbonyl and R₁₃ is a removable protecting group otherthan fluorenylmethoxycarbonyl, comprising reacting a compound of formulaVIII with a fluorenylmethoxycarbonyl donor compound, e.g.fluorenylmethoxycarbonyl-N-hydroxysuccinimide.

The invention will now be described with reference to the followingspecific embodiments, in which the following abbreviations are used:

Fmoc = fluorenylmethoxycarbonyl Boc = tert-butoxycarbonyl Cbo =carbobenzoxy (benzyloxycarbonyl) OSu = N-hydroxysuccinlmide HPTF =Heptane fraction JT = Jacket temperature HPLC = High performance liquidchromatography THF = Tetrahydrofuran TBME = Tert-butyl methyl ether DMF= Dimethylformamide

EXAMPLE 1 Preparation of 4-(diphenyl-hydroxy-methyl)-benzoic acid

1,4-dibromobenzene (47.2 g, 0.2 M) is added to THF (240 ml). The clearsolution is cooled to −65° C. A butyllithium solution (0.22 M, 94 ml ofa 20% solution in CHX) is added over 30 minutes.

After 5 minutes of stirring a solution of benzophenone (36.4 g, 0.2 M in180 ml THF) is added over 30 minutes (exothermic). The mixture isstirred for a further 30 minutes at −65° C. Then over 30 minutes thetemperature is raised to −10° C. and the solution is stirred at thistemperature for one hour.

The reaction mixture is then re-cooled to −65° C. Over 30 minutes abutyllithium solution (0.22 M, 94 ml of a 20% solution in cyclohexane)is added.

The resulting suspension is diluted with 200 ml THF. Then carbon dioxidegas is introduced over 90 minutes at 65° C. The temperature is raised to20° C. and the mixture stirred overnight. The mixture is then cooled to0° C. and an aqueous solution of ammonium chloride (120 ml of a 10%solution) is added over 30 minutes. 4-(diphenyl-hydroxy-methyl)-benzoicacid is formed at this stage.

The mixture is evaporated at 45° C. under a vacuum. The residue isadjusted to pH 4 with acetic acid and mixed with 400 ml H₂O. Extractionis performed with 2×150 ml ethyl acetate. The organic phases areextracted again with 100 ml water. The combined EST-phases are shakenwith a 10% aqueous potassium hydroxide solution (2×120 ml). The combinedaqueous phases are adjusted to pH 1-2 with hydrochloric acid at 20° C.and then extracted with 2×150 ml TBME. The combined TBME phases aremixed with 50 ml water and 50 ml saturated Na₂SO₄, dried with magnesiumsulphate and evaporated at 45° C. under vacuum to obtain a crudeproduct.

38.3 g crude product is dissolved in TBME (300 ml) at 40° C. The clearyellow solution is concentrated in a volume of 60 ml (240 ml TBMEdistilled off). The mixture is stirred for one hour at 40° C.(crystallisation). 50 ml HPTF is added, the mixture is cooled to 0° C.and stirred at 0° C. for 1 hour. Evaporating and washing with 2×15 mlheptane fraction and drying overnight at 45° C. under vacuum gives whitecrystals.

Attachment of 4(diphenyl-hydroxy-methyl)benzoic acid to a Solid Phase

15 g 4-(diphenyl-hydroxy-methyl)-benzoic acid with 7.54 ghydroxybenzotriazole is dissolved in 140 ml DMF by stirring for 15 min.15.3 ml di-isopropylcarbo-di-imide is added and the solution kept atroom temperature for 30 min. The solution is then stirred overnight atroom temperature in the presence of aminomethylated polystyrene. Afterwashing with DMF, methanol and THF the linker derivatised support isdried under vacuum.

EXAMPLE 2 Preparation of 4-(diphenyl-hydroxy-methyl)-benzoic acid(Alternative Method)

To 12 It TBME in a well dried 100 It Hastelloy-Reactor, 3.0 kgn-butyllithium (20% in cyclohexane; 9.37 mol) is added during a periodof 20 min, keeping the temperature at −5° C. (clear solution). During aperiod of 30 min 2.00 kg 1,4-dibromobenzene (8.48 mol) dissolved in 16 ITBME is added keeping the temperature between −50 and 0° C. The additioncontainer is rinsed with 3 I TBME.

After 30 min stirring at −5° C. a solution of 1.55 kg benzophenone (8.50mol) in 8 It TBME is added during a period of 20 min keeping thetemperature between −5° and 0° C. The addition container is rinsed with3 It TBME. A very small amount of a white solid is formed. After 15 minstirring at −5° C. a process control sample (HPLC 1) is taken. Thereaction mixture is stirred for further 25 min at −5° C. and warmed upto +25° C.

3.2 kg n-butyllithium (20% in cyclohexane; 10.00 mol) is added during aperiod of 25 min keeping the temperature between +25 and 27° C. Theaddition is slightly exothermic and the colour turned to slightly green.Some precipitate and froth are formed. After 20 min stirring a processcontrol sample is taken (HPLC 2). Depending on the result of HPLC 2further 0.3 kg n-butyllithium is added after stirring at +25° C. for 35min. After 15 min stirring a sample for process control is taken (HPLC3). The lines of the n-butyllithium addition are rinsed with 1.5 It TBMEand the reaction is cooled to −10° C.

1.99 kg dry ice (solid CO₂) is added portionwise during a period of 20min keeping the temperature between −10 and −5° C. The reaction isexothermic and a slightly yellow precipitate is formed. After 15 minstirring at −10° C. 11 I TBME are added and the reaction mixture iswarmed to 0° C. 5 I 18% aqueous hydrochloric acid is added during aperiod of 15 min keeping the temperature between 0° and +5° C. Theaddition is exothermic and the precipitate is dissolved (pH≦1).

The clear solution is transferred to a separation tank and the reactoris rinsed with 5 It TBME. After separation of the aqueous phase theorganic phase is washed with 20 it water. After separation of the twolayers the organic phase is extracted with 13 It 5% aqueous KOHsolution. The basic water phase is separated and the organic layer isextracted again with 13 It 5% aqueous KOH solution. The combined basicaqueous layers are transferred to the 100 It Hastelloy-Reactor. 22 I ofTBME and 6 I aqueous 18% hydrochloric acid are added over a period of 20min at a temperature between 0° and +5° C. The addition is exothermicand a white precipitate is formed but it dissolves again at a lowpH-value (pH≦1 after HCl-addition). The mixture is stirred during 10 minand transferred to a separation tank. The layers are separated and theaqueous phase is extracted again with 16 I TBME. After separation of thelayers the combined organic phases are concentrated at 500 mbar/45° C.JT to a volume of 4-5 I (32 I TBME are destined off) and seed crystalsare added. The temperature is raised up to 50° C. and 20 I HPTF areadded slowly with good stirring. The white precipitate is stirred for 2h at 50° C. JT. The jacked temperature is regulated at 0° C. andstirring is continued over night (16 h) letting cool down the suspensionto 0° C. The white suspension is filtered off and the reactor is rinsed5 times with 5 It of the mother liquor. The residue is dried at 45° C.JT under vacuum (≧10 mbar) to constant weight (over night).

EXAMPLE 3 Preparation of Fmoc-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OHstarting from Cbo-(2S,4R)-Pro(4-OH)—OMe

1. Dimethylaminopyridine (30.5 g, 250 mmol) andCbo-(2S,4R)-Pro(4-OH)—OMe (34.9 g, 125 mmol) are dissolved in toluene(870 ml). A solution of 4-nitrophenylchloroformate (31.5 g, 157 mmol) intoluene (206 ml) is added dropwise to this solution at 0° C. to 5° C.over 20 minutes and stirred for an additional 2 hours. This is followedby addition of a solution of Boc-ethylenediamine (80.1 g, 500 mmol) intoluene (205 ml) and stirring at ambient temperature for 12 hours. Asolution of concentrated sulfuric acid (43.7 g, 450 mmol) in water (873ml) is then added while maintaining a temperature of 20° C. to 25° C.The white suspension is filtered by suction and washed with toluene (30ml). The toluene phase is washed with water (450 ml), sodium carbonate(10% w/w, 450 ml) and three times with water (450 ml each). The toluenephase is azeotropically dried by distilling off 300 ml, which iscontinuously replaced by dry toluene (2×300 ml). Heptane (130 ml) isadded to the dry toluene solution at 50° C. and cooled to 0° C. over twohours. The precipitated product is filtered, washed two times withtoluene/heptane 1:2 v/v (70 ml), and dried at 50° C. under vacuum toleave Cbo-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OMe as a white solid.

2. Cbo-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OMe (20.0 g, 43.0 mmol) isdissolved in a 1:1 mixture of tetrahydrofuran and methanol (380 ml). A 1M sodium hydroxide solution (51.6 ml) is added and the resulting mixturestirred for 4 hours at ambient temperature. The mixture is adjusted topH 3 by adding sulfuric acid (50 ml, 1 M). Tetrahydrofuran and methanolare distilled off at 50° C. and 50 mbar until no further solventsdistil. The remaining milky solution is diluted with isopropyl acetate(113 ml) and water (57 ml), the phases are separated and the isopropylacetate phase is washed with sodium chloride solution (10%, 113 ml). Thesolvent is distilled off (50° C., 50 mbar) to yield a foam ofCbo-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH (19.8 g), which was usedwithout further purification in the next reaction.

3. Palladium on charcoal (10%, 1.94 g, 0042 mmol) is added to a solutionof Cbo-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH (19.4 g, 43.0 mmol) inisopropanol (350 ml) and water (37 ml). Hydrogen is bubbled through thismixture for 4 hours, the catalyst is filtered off, and the residue iswashed with a mixture of isopropanol (50 ml) and water (50 ml). Theisopropanol/water phase is azeotropically dried by distilling off ⅔ ofthe volume, which is continuously replaced by a toluene/isopropanolmixture (1:1 v/v). The remaining dry solution is concentrated in vacuoto dryness (50° C., 200 mbar) to leave(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH as a brownish solid, which wasused without further purification.

4. (2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH (5.0 g, 15 mmol) is dissolvedin a mixture of water (25 ml) and triethylamine (1.5 g, 15 mmol) at 40°C. A solution of Fmoc-OSu (4.65 g, 14 mmol) in acetonitrile (25 ml) isadded to the clear solution over 30 minutes and stirred for 2 hours.Then the reaction mixture is adjusted to pH 3 with hydrochloric acid (1m, 13 ml) and stirred for a further hour. Acetonitrile is distilled off(40° C., 80 mbar) and replaced by isopropyl acetate, affording atwo-phase mixture. The lower aqueous phase is separated off, whilst theremaining organic layer is washed with water and distilled two timeswith replacement with isopropylacetate and then concentrated to abrownish foam. This foam is dissolved in isopropylacetate (25 ml) andadded dropwise to heptane (200 ml) whereby the product is precipitated.The solid is filtered, washed with isopropylacetate/heptane and dried invacuo at 40° C. to leave Fmoc-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH.

EXAMPLE 4 Preparation of Fmoc-(2S,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OHStarting from Cbo-(2S,4R)-Pro(4-OH)—OBzl

The synthesis of Cbo-(2S,4R)-Pro(4-OH)—OBzl is described in T. Makoto,H. Guoxia, V. J. Hruby, J. Org. Chem. 2001, 66, 1038-1042. The processof example 3 is repeated, but using Cbo-(2S,4R)-Pro(4-OH)—OBzl in placeof Cbo-(2S,4R)-Pro(4-OH)—OMe and performing steps 1, 3 and 4 only(omitting step 2).

EXAMPLE 5 Preparation of Fmoc-(2R,4R)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH

The process of example 3 or example 4 is repeated but usingCbo-(2R,4R)-Pro(4-OH)—OMe or Cbo-(2R,4R)-Pro(4-OH)—OBzl in place ofCbo-(2S,4R)-Pro(4-OH)—OMe or Cbo-(2S,4R)-Pro(4-OH)—OBzl.

EXAMPLE 6 Preparation of Fmoc-(2S,4S)-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH

The process of example 3 or example 4 is repeated but usingCbo-(2S,4S)-Pro(4-OH)—OMe or Cbo-(2S,4S)-Pro(4-OH)—OBzl in place ofCbo-(2S,4R)-Pro(4-OH)—OMe or Cbo-(2S,4R)-Pro(4-OH)—OBzl.

1-4. (canceled)
 5. A compound of formula VI in free or salt form

wherein R₃, R₄ and R₅ are each independently hydrogen or one or moresubstituents attached to each benzene ring, and are selected fromhydroxy, amino, C₁₋₁₀-alkyl, C₁₋₁₀-alkoxy, C₁₋₁₀-alkylamino,di-C₁₋₁₀-alkylamino, carbamoyl, C₁₋₁₀-alkylcarbamoyl,di-C₁₋₁₀-alkylcarbamoyl, halo-C₁₋₁₀-alkyl, halogeno or nitro, optionallyprotected by a removable protecting group; R₉ is —OH, —OM or —OMX, whereM is metal and X is a nucleophilic substituent; and R₁₀ is -M or -MX,where M is metal and X is a nucleophilic substituent. 6-10. (canceled)